Mapping the Distribution of Conformational Information
Throughout a Protein Sequence
Leopoldo G. Gebhard1,2, Valeria A. Risso1,2, Javier Santos1,2
Raul G. Ferreyra1,2, Martı ´n E. Noguera1and Mario R. Erma ´cora1,2*
1Departamento de Ciencia y
Tecnologı ´a, Universidad
Nacional de Quilmes, Roque
Sa ´enz Pen ˜a 180, (1876) Bernal
Buenos Aires, Argentina
2Consejo Nacional de
Investigaciones Cientı ´ficas y
Te ´cnicas,Rivadavia 1917 (1033)
Ciudad Auto ´noma de Buenos
The three-dimensional structure of protein is encoded in the sequence, but
the identity of those that are structure-determining is elusive. By circular
permutation and terminal deletion, we produced and purified 25 Bacillus
licheniformis b-lactamase (ESBL) variants that lack 5–21 contiguous residues
each, and collectively have 82% of the sequence and 92% of the non-local
atom–atom contacts eliminated. Circular dichroism and size-exclusion
chromatography showed that most of the variants form conformationally
all populate, to a greater or lesser extent, conformations with the essential
local information and modular organization can impart most of the tertiary
fold specificity and cooperativity.
q 2006 Elsevier Ltd. All rights reserved.
Keywords: protein folding; sequence patterns; conformational information;
folding code; folding units
The conformation of a protein is encoded in the
sequence,1and unraveling the folding code has
become one of the greatest challenges to contem-
porary science. A major difficulty in solving this
problem is the lack of obvious correspondence
between sequence and three-dimensional structure:
proteins tolerate a surprisingly large number of
there are several examples of
non-homologous proteins belonging to the same
structural class;9proteins with highly similar
sequence but different fold have been created;10
and identical sequences of 8–11 residues adopt
different structures in different proteins.11,12
To elucidate the logic of folding it is necessary to
establish if there are structure-determining sequence
is the most powerful predictor of tertiary structure,
exhaustive examination of natural and experimen-
tally mutated proteins has so far failed to identify
prototypic sequence signatures characteristic of each
suggests that the amino acid alphabet may be
and hydrophilic residues may promote native-like
folding and efficient core packing.13Furthermore,
many residues may carry no global conformational
information, as can be concluded from statistical
analyses,14and the observation that some protein
fragments do achieve native-like folds.15–25
With the above considerations in mind, we
devised an experimental procedure for the identifi-
cation of sequence segments that may carry essential
involves terminal truncation of circularly permuted
proteins. The approach allows sliding a deletion
window along the chain, and the resulting abridged
variants reveal the structural consequences of
lacking specific parts of the sequence. Unlike
conventional site-specific mutagenesis, this segmen-
tal deletion can be used to switch off interactions
involving main chain atoms.
As a proof of principle, we performed segmental
deletion on the two-domain, 264-residue protein
Bacillus licheniformis b-lactamase27(ESBL; Figure 1).
The first domain, which is made of middle-chain
residues, exhibits a central a-helix surrounded by a
0022-2836/$ - see front matter q 2006 Elsevier Ltd. All rights reserved.
Abbreviations used: BP, benzyl penicillin; cp, circularly
permuted; ESBL, B. licheniformis exo small b-lactamase;
NC, nitrocefin; SEC, size-exclusion chromatography.
E-mail address of the corresponding author:
doi:10.1016/j.jmb.2006.01.095J. Mol. Biol. (2006) 358, 280–288
and 310helices. The second domain contains a five-
stranded, antiparallel b-sheet plus the N and
C-terminal a-helices. The general folding properties
of ESBL have been characterized.25,28
A total of 25 ESBL variants carrying deletions that
cover almost the entire sequence were prepared and
purified. In vitro enzymic activity was found to be a
convenient probe for the detection of native-like
structure in the presence of vast amounts of
misfolded protein. Circular dichroism (CD) spec-
complements to further assess the structural con-
sequences of the deletions. Contrary to expectation,
the results suggest that no part of ESBL sequence
carries essential conformational information. This
which the amino acid positions are only locally
coupled and the protein matrix is built by the
assembly of self-organizing modules.
Expression and global properties of ESBL
Combining circular permutation and terminal
deletion, we produced ESBL variants lacking
fragments of 5–21 residues along the sequence up
the nomenclature of ESBL variants is explained in
Table 1). To represent the structural consequences of
the deletions, all contacts between residues sepa-
rated in sequence by four or more positions were
calculated from the crystallographic model (PDB
entry 4blm). With a 4.5 A˚cutoff, there are 2897
atom–atom contacts involving 536 residue–residue
interactions. The distribution of contacts removed
by each truncation is shown in Figure 3. Typically,
in the network of tertiary contacts, each mutant
lacks one node that ramifies to three to five elements
of distant tertiary structure and establishes more
than 100 atom–atom contacts. Summing over all the
variants, 92% of the non-local atom–atom contacts
The ESBL variants could be expressed with good
yields (10–300 mg/l of culture) and purified to
homogeneity. In roughly half of the cases, signifi-
cant amounts of insoluble product accumulated.
Interestingly, the variants with deletions involving
the first 37 or the last 41 amino acid positions were
the most insoluble.
In the far-UV region, nearly all ESBL variants
exhibit CD spectra with native-like shape and
moderate variations in signal intensity likely to
arise from the intrinsic contribution of the deleted
residues tothe native
Cp276\282, the only exception to this trend,
shows a spectrum suggestive of a large change
in secondary structure. In the near-UV, the
changes between mutants are more conspicuous:
cp64, cp255, and cp267\276, have CD spectra
with native-like shape and intensity, several have
native-like shapes but decreased intensities; and
nearly half show spectra with little or no
The optical results show that the average
secondary structure content of ESBL is remarkably
resistant to segmental deletion. In contrast, the
average tertiary structure of most ESBL variants
was disrupted significantly, which is consistent
with the observed tendency of this protein to
populate partially folded states.25,28
Molecular size and aggregation state analysis by
size-exclusion chromatography (SEC) also revealed
dissimilar behaviors: six variants are exclusively
monomeric and as compact as the wild-type protein
(cp217\226, cp163\178, cp227\231, cp267\276,
cp64, cp255); four yield compact monomeric species
along with different amounts of aggregates and
Figure 1. Molecular model of ESBL. The a and aCb
domains are shown in green and blue, respectively. The
residues that participate in catalysis are indicated. The
Figure was prepared using Swiss-PDBViewer (http://
www.expasy.org/spdbv/) and POV-Raye (http://www.
Figure 2. Sequence coverage. Bars encompass the residues deleted in each ESBL variant. Numbering is that proposed
by Ambler.56Asterisks show catalytically important residues.
Conformational Information Throughout Protein Sequence
cp236\245, D287\295), and the remaining populate
dimeric and aggregated states only (not shown).
The specific effects of the pentaglycine connection
between residues 27 and 295 on ESBL conformation
were assessed by examining cp64 and cp255.
Judging from the SEC and CD data, the confor-
mation of these two full-length, circularly permuted
variants is identical with that of the wild-type
protein. This was confirmed by the catalytic
parameters (Table 1). Thus, the common penta-
glycine connection per se does not alter the fold of
ESBL significantly and can be considered neutral as
regards to conformational information.
Optical and hydrodynamic data concurrently
show that the abridged variants tend to form
complex mixtures in which altered conformations
predominate. Therefore, to assess whether they
retain the capacity to fold to native-like states, we
resorted to a more sensitive and specific probe.
Catalysis as a probe for native folding
Catalysis depends strongly on conformational
details,29and inactivation precedes or accompanies
early conformational changes during protein
unfolding.30Moreover, catalytic activity can moni-
tor folding with very high levels of sensitivity and
specificity because the signal proceeds only from
the native conformation. This makes feasible the
detection of traces of native fold in preparations
containing large amounts of unfolded and/or
In ESBL, residues involved directly in catalysis
and/or substrate binding31,32are particularly well
suited to monitor the relative spatial arrangement
and conformational integrity of the two protein
domains (Figure 1). In the a domain, the Ogatom of
S70 is activated for nucleophilic attack on the lactam
carbonyl group to yield an acyl enzyme intermedi-
ate, and E166 participates as a general base in the
subsequent deacylation step. The role of K73 is in
facilitating the protonation of the lactam leaving
group in acylation and of the Ogleaving group in
deacylation. This is most likely achieved through
electrostatic effects on other active site residues, like
S130, and by the transfer of a proton to E166 via a
fixed water molecule. N132 and N170 contribute to
substrate binding by making two hydrogen bonds
to the carbonyl amide group on lactam C10.
On the other hand, several aCb-domain residues
participate in the catalytic mechanism. Main-chain
atoms of A237 establish two hydrogen bonds to C6
and C7 lactam substituents and, along with the
three hydrogen bonds joining R244 and T235 to the
carboxylate at C2, ensure that the substrate covers
the Ser70 Ogwith the target carbonyl group placed
properly on the enzyme oxyanion hole. There,
nitrogen atoms from A237 and S70 polarize the
carbon–oxygen bond involved in the formation of
the tetrahedral intermediate. In addition, K234
makes a contribution to the active site hydrogen
Table 1. Catalytic parameters of ESBL variants
Variant Deletion (residues)kcata(sK1)
aAverages of two, three or four independent measurements are reported. The standard deviation of kcatand Kmwas !20%.
bESBL variants names are as follows: the prefix cp indicates circular permutation; D is used for abridged variants with wild-type
connectivity; two numbers separated by\identify the N and C terminus of the removed sequences; single numbers are used to identify
the new N terminus of full-length, circularly permuted variants; amino acid numbering is the consensus for class A b-lactamases.56
Conformational Information Throughout Protein Sequence
bond network and, along with S130 and T235, fixes
the substrate, through lacta N4 and the carboxylate
group at C3, in the right position for catalysis.
Furthermore, the precise three-dimensional regis-
ter of active site residues depends on the integrity of
several elements of secondary and tertiary structure
from both domains: (a) S70 and K73 monitor a-helix
71–83, the central, buried ”master column” of the
a-domain;(b) K234, T235, A237, and R244 survey the
central, five-stranded b-sheet in the aCb domain; (c)
E166 and N170 report on the conformation of the U
loop; and (d) S130 and N132 sense the loop
connecting helices 120–128 and 132–140.
The entangled nature of the active site and the
number and the intricacy of its geometric con-
straints indicate that the conformational integrity of
most of the two domains is required to sustain
catalysis, and that ESBL can be enzymically active
only if folded in a native-like fashion. Further
evidence confirms that catalytic activity can be
used to monitor the native fold of ESBL as a whole:
(a) spectroscopic, hydrodynamic, and chemical
modification experiments demonstrated that the
ESBL domains do not unfold independently during
urea-induced unfolding experiments at equilibrium
(J.S. et al., unpublished results); and (b) isolated
recombinant ESBL fragments corresponding to the
a and aCb domains have a strong tendency to
aggregate when refolded from urea solutions and
are enzymically inactive (V.A.R. & M.R.E., unpub-
We considered how to distinguish true enzymic
activity from uncatalyzed hydrolysis or from the
reaction with isolated nucleophiles. Fortunately, an
enlightening guide to enzymic activity thresholds
could be found in previous works.3,33At micro-
molar concentrations, compared with uncatalyzed
reactions, typical enzymes increase reaction rates by
2–17 orders of magnitude.
In good agreement with previous reports,34we
determined that the uncatalyzed first-order rate
for benzyl penicillin (BP) hydrolysis in buffer A
(100 mM sodium phosphate, (pH 7.0 at 25 8C)) is
2.6!10K7sK1. Also, we found that the rate in the
presence of lysozyme or bovine serum albumin
(BSA) is similar to that in buffer alone (Table 2).
These two proteins are large and diverse enough to
represent typical protein surfaces.
We did not use active site mutants of ESBL as
negative controls of enzymic activity because
mutants lacking “essential” catalytic residue may
still perform catalysis through alternative mechan-
Figure 3. Pairs of residues that contact each other in the
native structure of ESBL. Units are residue number.
Residues separated by four or more positions in sequence
are considered a pair if they make contact through atoms
separated by less than 4.5 A˚. Colors indicate how many
atom–atom contacts each pair of residues establishes. Red
bars indicate the segment of sequence removed in each
Figure 4. Far-UV and near-UV
spectra. The variants were refolded
by dialysis against buffer A before
analysis (see Methods).
Conformational Information Throughout Protein Sequence
variant of ESBL, which lacks the nucleophile that
an increased rate of substrate hydrolysis compared
with the reaction in buffer alone.37Similarly, S70A
activity (Table 2).38Instead of active site mutants, we
assayed ESBL(Cya)2, a variant of ESBL with sulfonic
acid at positions 126 and 265, which is intended to be
permanently unfolded (J.S. et al., unpublished
results). As expected, BP hydrolysis in the presence
of ESBL(Cya)2showed no Michaelian behavior and
yielded a linear plot up to 2 mM substrate (Figure 5).
From the slope of the plot, an apparent second-order
rate constant of 0.44 MK1sK1was calculated
Since the rates of BP hydrolysis by ESBL(Cya)2,
lysozyme, and BSA are similar, it can be concluded
safely that the rate of uncatalyzed hydrolysis, either
by solvent or by protein residues in unspecific
arrays, is not greater than 0.44 MK1sK1. On the
other extreme, we found that kcat/Kmfor wild-type
ESBL is 1.8!107MK1sK1. These values set a
natural scale for BP hydrolysis spanning eight
orders of magnitude.
Lastly, the catalytic parameters for all the
abridged b-lactamases were determined from
Michaelis–Menten curves (Table 1; Figure 5). To
judge the results properly, the following consider-
ations should be taken into account. (a) Reported
kcat/Kmvalues are lower limits because most of the
lactamase preparations contain significant amounts
of inactive protein. (b) Although only results for BP
hydrolysis are shown, specific activity toward BP
and nitrocefin (NC) was comparable. (c) Cross-
contamination between ESBL variants was avoided
by using new chromatographic matrix and buffers
in each purification. (d) A dummy chromatography
of a control homogenate of cells expressing an
unrelated protein demonstrated that no enzymi-
cally active contaminants co-purify with ESBL (not
shown). (e) The rate of BP hydrolysis by a raw
control bacterial homogenate was similar to that
promoted by buffer A alone (not shown).
Considering both the activity of existing enzymes
and the activity scale established above, it seems
reasonable to assume that true lactamase-catalyzed
reactions will have second-order rates at least two
proteins. By this criterion, we found that all the
truncated variants prepared for this work display
genuine enzymic activity (Figure 6). Since the
observed enzymic activity is unlikely to be the result
of a novel ESBL fold induced by the truncations, and
that all the preparations of truncated ESBL variants
The ESBL folding code is robust
Most of the circularly permuted and abridged
ESBL variants prepared for this work exhibit
Table 2. Reference values for b-lactamase activity
Reagent Rate (MK1sK1) Typical velocitya(MK1sK1)Ratiob
aCalculated as vZ[Reagent][BP]k, where [Reagent]Z1!10K6M, [BP]Z2!10K3M and k is the second-order rate; or, for reaction
buffer, as vZ[BP]k, where k is the apparent first-order rate.
bQuotient of reactive group velocities and reaction buffer velocity.
cIn water, at 30 8C.34
eCalculated by measuring hydrolysis rate as a function of protein concentration.
fStreptomyces albus G b-lactamase.38
Figure 5. Examples of reaction rate against substrate
concentration curves. The three circularly permuted and
truncatedESBL variants with the lowest kcat/Kmvaluesare
shown (see Table 1) along with ESBL(Cya)2, the unfolded
negative control for enzymic catalysis (see the text).
Conformational Information Throughout Protein Sequence
conformational heterogeneity, a pronounced ten-
dency to form aggregates, and low refolding yield,
but all of them retain the ability to fold into a native-
like, enzymically active conformation. Since these
variants lack segments of sequence, significant
amount of specific tertiary contacts, and wild-type
backbone extremes, this implies a conformational
code that is robust against elimination of infor-
mation, changes in chain connectivity, and altera-
tion in the relative location of information units.
Moreover, since the applied deletion scheme
perturbs the entire network of long-range, native
contacts, it follows that the instructions for ESBL
structure cannot be in the form of a unique pattern
of essential interactions distributed along the
sequence. Likewise, since atom–atom contacts are
a consequence of the type of residue at each
sequence position, the three-dimensional specificity
must be conveyed independently of any particular
detail of the sequence.
It would not be possible to reach the above
conclusions from residue substitution experiments
or evolutionary variability analysis alone because:
(a) mutations modify but do not eliminate the
conformational information at a given sequence
position; (b) sequence positions that are highly
tolerant to amino acid substitution still may carry
significant non-local conformational information;
(c) invariant or highly conserved residues may be
non-essential for the global conformation and
reflect only local structural constraints; and (d) the
combinatory nature of compensating mutations
precludes an exhaustive experimental exploration
of all possible sequences related to the same fold.
The observed robustness of ESBL folding code
imposes restrictions on the arrangement of confor-
mational information over the length of the
sequence. If robustness were achieved by redun-
dancy, there would be sets of complete folding
instructions stored in different sites along the chain.
However, using Radar,39we found no internal
homology in ESBL and, thus, the existence of
redundant blocks of information in it seems
A more parsimonious way to explain ESBL
robustness is modular construction. In the hier-
archic model of folding,40the structure determi-
nants are local in sequence, long-range contacts
only consolidate pre-existing local structure, and
non-local, three-dimensional specificity results
from the binding of independently ordered mod-
ules. By definition, hierarchic models are tolerant to
segmental deletion, because the associated confor-
mational disruption can be circumscribed to par-
ticular modules without precluding the folding and
assembly of the others.
The robustness of ESBL code is more difficult to
justify by assuming models of folding that propose
the growth of tertiary structure by concerted
consolidation of secondary and tertiary interactions
from a nucleation site.41Segment deletion elimin-
ates simultaneously hundred of long-range con-
tacts, including those established by main chain
atoms (Figure 3). If the folding of ESBL were a
process dependent on the specific and concerted
formation of a significant number of tertiary
contacts, the deletion scheme would have identified
several variants incapable of folding. Yet, formally,
the possibility exists that multiple alternative routes
for folding may allow these mutants to compensate
for the loss of any particular set of tertiary contacts.
This, however, would be possible only if ESBL
contained a large number of potential nucleation
To our best knowledge, the results hereby
reported for ESBL constitute the first verification
over an entire sequence of the robustness of the
folding code. Since ESBL is representative of
medium-sized and relatively complex protein
architecture, it remains to be seen if the folding
code is also robust in small, single-domain proteins
with a simpler design. Nevertheless, the fact that
diverse abridged proteins showing native-like
folding could be produced suggests that ESBL is
not an exception.15–24Admittedly, there are many
reports of folding impairment as a consequence of
circular permutation, deletions, and even point
mutations. From systematic circular permutation,
Iwakura et al.42have concluded that DHFR contains
ten sequential elements essential for folding, which
is seemingly at odds with the picture that emerges
from the experiments with ESBL. However, as
discussed above, to ponder on the evidence, it is
important to separate conceptually the sequence
changes that may cause instability, aggregation,
affect kinetics, or otherwise reduce the yield of
native structure, from those that may eliminate
altogether the possibility of native fold.43If what
is under examination is the specificity of folding,
then negative results should be considered incon-
clusive unless the possibility of native-like folding,
even at trace levels, is explicitly discarded. Failure
to do so may still allow learning about the structural
determinants of folding stability and efficiency, but
will not clarify the logic of the folding code.
Figure 6. Catalytic activity. The dotted line is the
assumed threshold for genuine enzymic activity (see the
text and Tables 1 and 2).
Conformational Information Throughout Protein Sequence
Concerning this, we believe that many of the
studies reporting folding incapacity have not been
properly designed to detect the presence of small
quantities of natively folded molecules among a
large excess of misfolded products.
A revealing example furtherhighlightsthe needto
1–103 fragment of the 149-residue protein staphylo-
coccal nuclease appears unstructured by NMR
analysis. However, if stabilized by mutations V66L
and G88V, this fragment adopts a stable tertiary
in the X-ray structure of the parent protein.44Thus, it
in the absence of the 46 C-terminal residues.
The modular design of protein structure
The ability of the permutation–truncation
approach to switch off elements of tertiary structure
is perhaps its most interesting feature. Circularly
permuted, truncated proteins can be considered as
designed folding intermediates, in which specific
elements of tertiary structure are unfolded by
elimination of sequence. In this regard, as probes
for folding reach residue resolution and more
detailed analysis are performed, the hidden multi-
state nature of protein molecules becomes more
apparent. The picture that emerges from equili-
brium studies by NMR spectroscopy in conjunction
with hydrogen exchange shows elements of native
structure whose transient unfolding does not
depend on global unfolding.45,46Thus, our results
and the latest direct structural information concur
to challenge established ideas on long-range
cooperativity of protein structure.
Switching off structural elements by sequence
deletion is also conceptually close to the compu-
tational approach to macromolecular equilibrium
advocated by Hilser and colleagues.47Those
authors proposed that the native state is a statistical
ensemble of conformations that originate from the
existence of local unfolding transitions throughout
most of the protein molecule. Their calculations,
based on segmental unfolding, achieved remark-
able success in predicting experimental hydrogen-
exchange protection factors. From the informational
standpoint, the experiments with ESBL and the
independent evidence mentioned above support
the contention that no essential subset of tertiary
interaction encodes the native structure, and that
the native protein matrix is an intrinsically modular
construction that is determined locally.
Since its introduction,48,49the concept of modular
protein design has been the inspiration for many
investigations. In particular, several authors have
undertaken the characterization of autonomous
folding units by a variety of approaches.50–53By
definition, folding units are made of contiguous
residues and contain elements of secondary and
local tertiary structure. In solution, the predominant
conformation of these units would be similar to that
acquired in the native protein; but it is possible that
the mutually stabilizing association between units
may select alternative, less-well populated confor-
mations. Thus, there would be no formal contra-
conformational ambiguity. In agreement with this
idea, prediction methods in which structure
sampled by local sequence is approximated by the
distribution of prototype segments structures in
data banks achieved impressive success.54
In sum, the present results demonstrate that the
ESBL fold is resistant to sequence deletion to an
extent that precludes the existence of discrete
patterns of amino acid positions essential for
specifying the overall three-dimensional structure.
Thus, no particular network of inter-atomic inter-
ESBL is congruent with experimental evidence and
theoreticalconsiderations suggestingthatlocal infor-
the tertiary fold specificity. If this is a property of
proteins in general, theoretical and empirical studies
on protein folding will be bolstered by a huge
BP and NC were from Sigma (St. Louis, Missouri) and
Calbiochem (La Jolla, CA), respectively. Protein purity
was assessed bySDS-PAGE. CD andSEC were carried out
Preparation of ESBL variants
Site-specific and abridged mutants of ESBL with wild-
type chain connectivity were prepared essentially as
described.25The general strategy for circular permutation
was that applied to DHRF by Iwakura et al.42Briefly, a
repeat offive Gly codons was ligated between two copies
of the DNA coding for ESBL, andthe construction was the
template for PCR amplification of the segmentally deleted
variants. Primers included restriction sites for cloning
into pET9a (Novagen, Madison, USA). The absence of
unwanted mutations was confirmed by DNA sequencing
(IIB, UNSAM, Argentina). To prepare ESBL(Cya)2,
S126C–S265C ESBL, which has 80% of the specific activity
of wild-type ESBL and one inaccessible cysteine residue
per protein domain (J.S. et al., unpublished results), was
oxidized with performic acid,55dialyzed against buffer A,
and freed from aggregates by filtration through 0.1 mm
pore size filters.
Escherichia coli BL21 (DE3) cells transformed with
pET9a carrying the modified ESBL genes were cultured
in Luria-Bertani medium at 37 8C to A600nmw1.0, then
induced with 1 mM IPTG (3 h), and finally harvested by
centrifugation. ESBL, S126C–S265C ESBL, and circularly
permuted 255 ESBL (cp255) were purified as described.28
The variants that accumulated with good yield in
inclusion bodies were purified as described.25The rest,
which were soluble but unstable, conformationally
altered, or prone to aggregation, could not be purified
by conventional protocols. For these variants, we applied
the following purification procedure. First, cell homogen-
ates were subjected to a short incubation (10 min, 20 8C)
Conformational Information Throughout Protein Sequence
with 5 M urea, 5 mM glycine, 25 mM phosphoric acid
(pH 3.5). We discovered serendipitously that this
treatment causes bulk precipitation of most bacterial
proteins and leaves in solution the unstably folded ESBL
variants close to purity. Second, the proteins so isolated
were subjected to ionic exchange chromatography under
denaturing conditions.25,28In all cases, protein refolding
was done at 4 8C by dialysis against 50 mM sodium
phosphate (pH 7.0), and particulate material was
eliminated by centrifugation.
Substrate hydrolysis was monitored by UV absorption
at 240 nm (BP; D3Z570 MK1cmK1) or 486 nm (NC; D3Z
20000 MK1cmK1) with a Shimadzu UV-160A spectropho-
tometer (Shimadzu, Japan). All the assays were per-
formed at 25 8C in buffer A. Reactions were initiated by
adding 50 ml of 0.01–25 mM b-lactamase to 450 ml of
substrate (2.0 mM BP or 0.1 mM NC). Initial rates were
calculated from the change in absorbance that ensued
from the consumption of no more than 10% of the
substrate. kcat and Km were calculated by fitting the
Michaelis–Menten equation to velocities at various
concentrations of BP. Proteins unrelated to b-lactamase
(0.05–5.0 mM) were assayed. Non-enzymic hydrolysis of
BP was determined by incubating the antibiotic in buffer
A (3 days; at 25 8C in the dark). Two, three or four
independent measurements were performed for each
protein and controls.
We thank Professor Anthony Fink for the
lactamase gene and many suggestions on this
protein. This work was supported by grants from
CONICET, UNQ, and ANPCyT.
1. Anfinsen, C. B. (1973). Principles that govern the
folding of protein chains. Science, 181, 223–230.
2. Palzkill, T. & Botstein, D. (1992). Probing beta-
lactamase structure and function using random
replacement mutagenesis. Proteins: Struct. Funct.
Genet. 14, 29–44.
3. Axe, D. D., Foster, N. W. & Fersht, A. R. (1998). A
searchfor single substitutions
enzymatic function in a bacterial ribonuclease.
Biochemistry, 37, 7157–7166.
4. Wen, J., Chen, X. & Bowie, J. U. (1996). Exploring the
allowed sequence space of a membrane protein.
Nature Struct. Biol. 3, 141–148.
5. Rennell, D., Bouvier, S. E., Hardy, L. W. & Poteete,
A. R. (1991). Systematic mutation of bacteriophage T4
lysozyme. J. Mol. Biol. 222, 67–88.
6. Matthews, B. W. (1993). Structural and genetic
analysis of protein stability. Annu. Rev. Biochem. 62,
7. Gassner, N. C., Baase, W. A. & Matthews, B. W. (1996).
A test of the “jigsaw puzzle” model for protein
folding by multiple methionine substitutions within
the core of T4 lysozyme. Proc. Natl Acad. Sci. USA, 93,
8. Miller, O. J. & Dalby, P. A. (2004). Exposing
relationships using directed evolution. Trends Biotech-
nol. 22, 203–205.
9. Orengo, C. A., Jones, D. T. & Thornton, J. M. (1994).
Protein superfamilies and domain superfolds. Nature,
10. Alexander, P. A., Rozak, D. A., Orban, J. & Bryan, P. N.
(2005). Directed evolution of highly homologous
proteins with different folds by phage display:
implications for the protein folding code. Biochemistry,
11. Sudarsanam, S. (1998). Structural diversity of sequen-
tially identical subsequences of proteins: identical
octapeptides can have different conformations. Pro-
teins: Struct. Funct. Genet. 30, 228–231.
12. Minor, D. L., Jr & Kim, P. S. (1996). Context dependent
secondary structure formation of a designed protein
sequence. Nature, 380, 730–734.
13. Moffet, D. A. & Hecht, M. H. (2001). De novo proteins
14. Rackovsky, S. (1993). On the nature of the protein
folding code. Proc. Natl Acad. Sci. USA, 90, 644–648.
15. Rico, M., Jimenez, M. A., Gonzalez, C., De Filippis, V.
& Fontana, A. (1994). NMR solution structure of the C
terminal fragment 255 316 of thermolysin: a dimer
formed by subunits having the native structure.
Biochemistry, 33, 14834–14847.
16. Patrick, W. M. & Blackburn, J. M. (2005). In vitro
selection and characterization of a stable subdomain
of phosphoribosylanthranilate isomerase. FEBS J. 272,
17. Trevino, R. J., Gliubich, F., Berni, R., Cianci, M.,
Chirgwin, J. M., Zanotti, G. & Horowitz, P. M. (1999).
NH2-terminal sequence truncation decreases the
stability of bovine rhodanese, minimally perturbs its
crystal structure, and enhances interaction with
GroEL under native conditions. J. Biol. Chem. 274,
(2001). Conformational characterization of designed
minibarnase. Biopolymers, 58, 260–267.
19. De Sanctis, G., Falcioni, G., Polizio, F., Desideri, A.,
Giardina, B., Ascoli, F. & Brunori, M. (1994). Mini
myoglobin: native like folding of the NO derivative.
Biochim. Biophys. Acta, 1204, 28–32.
20. Kim, K., Cistola, D. P. & Frieden, C. (1996). Intestinal
fatty acid-binding protein: the structure and stability
of a helix-less variant. Biochemistry, 35, 7553–7558.
21. Rose, T., Brune, M., Wittinghofer, A., Le Blay, K.,
Surewicz, W. K., Mantsch, H. H. et al. (1991).
Structural and catalytic properties of a deletion
derivative (delta 133 157) of Escherichia coli adenylate
kinase. J. Biol. Chem. 266, 10781–10786.
22. Chamberlain, A. K., Fischer, K. F., Reardon, D.,
Handel, T. M. & Marqusee, A. S. (1999). Folding of
an isolated ribonuclease H core fragment. Protein Sci.
23. Searle, M. S., Zerella, R., Williams, D. H. & Packman,
L. C. (1996). Native-like beta-hairpin structure in an
isolated fragment from ferredoxin: NMR and CD
studies of solvent effects on the N-terminal 20
residues. Protein Eng. 9, 559–565.
24. Tasayco, M. L. & Carey, J. (1992). Ordered self-
assembly of polypeptide fragments to form nativelike
dimeric trp repressor. Science, 255, 594–597.
25. Santos, J., Gebhard, L. G., Risso, V. A., Ferreyra, R. G.,
Rossi, J. P. & Erma ´cora, M. R. (2004). Folding of an
abridged beta-lactamase. Biochemistry, 43, 1715–1723.
Conformational Information Throughout Protein Sequence
26. Clerico, E. M., Peisajovich, S. G., Ceolin, M., Download full-text
Ghiringhelli, P. D. & Erma ´cora, M. R. (2000).
Engineering a compact non-native state of intestinal
fatty acid-binding protein. Biochim. Biophys. Acta,
27. Moews, P. C., Knox, J. R., Dideberg, O., Charlier, P. &
Fre `re, J. M. (1990). Beta-lactamase of Bacillus licheni-
formis 749/C at 2 A˚resolution. Proteins: Struct. Funct.
Genet. 7, 156–171.
28. Frate, M. C., Lietz, E. J., Santos, J., Rossi,J. P., Fink, A. L.
& Erma ´cora, M. R. (2000). Export and folding of signal-
sequenceless Bacillus licheniformis beta-lactamase in
Escherichia coli. Eur. J. Biochem. 267, 3836–3847.
29. Fersht, A. (1999). Structure and Mechanism in Protein
Science: A Guide to Enzyme Catalysis and Protein Folding.
Freeman, New York.
30. Tsou, C. L. (1995). Inactivation precedes overall
molecular conformation changes during enzyme
denaturation. Biochim. Biophys. Acta, 1253, 151–162.
31. Fonze, E., Vanhove, M., Dive, G., Sauvage, E., Frere,
J. M. & Charlier, P. (2002). Crystal structures of the
Bacillus licheniformis BS3 class A beta-lactamase and of
the acyl-enzyme adduct formed with cefoxitin.
Biochemistry, 41, 1877–1885.
32. Lietz, E. J., Truher, H., Kahn, D., Hokenson, M. J. &
Fink, A. L. (2000). Lysine-73 is involved in the
acylation and deacylation of beta-lactamase. Biochem-
istry, 39, 4971–4981.
33. Radzicka, A. & Wolfenden, R. (1995). A proficient
enzyme. Science, 267, 90–93.
34. Llina ´s, A., Vilanova, B., Frau, J., Mun ˜oz, F., Donoso, J.
& Page, M. I. (1998). Chemical reactivity of penicillins
and cephalosporins. Intramolecular involvement of
the acyl-amido side chain. J. Org. Chem. 63, 9052–9060.
35. Peracchi, A. (2001). Enzyme catalysis: removing
chemically ‘essential’ residues by site-directed muta-
genesis. Trends Biochem. Sci. 26, 497–503.
36. Carter, P. & Wells, J. A. (1988). Dissecting the catalytic
triad of a serine protease. Nature, 332, 564–568.
37. Hokenson, M. J., Cope, G. A., Lewis, E. R., Oberg, K. A.
& Fink,A.L. (2000).Enzyme-induced strain/distortion
in the ground-state ES complex in beta-lactamase
catalysis revealed by FTIR. Biochemistry, 39, 6538–6545.
38. Jacob, F., Joris, B. & Frere, J. M. (1991). Active-site
serine mutants of the Streptomyces albus G beta-
lactamase. Biochem. J. 277, 647–652.
39. Heger, A. & Holm, L. (2000). Rapid automatic
detection and alignment of repeats in protein
sequences. Proteins: Struct. Funct. Genet. 41, 224–237.
40. Fitzkee, N. C., Fleming, P. J., Gong, H., Panasik, N., Jr,
Street, T. O. & Rose, G. D. (2005). Are proteins made
from a limited parts list? Trends Biochem. Sci. 30, 73–80.
41. Daggett, V. & Fersht, A. R. (2003). Is there a unifying
mechanism for protein folding? Trends Biochem. Sci.
42. Iwakura, M., Nakamura, T., Yamane, C. & Maki, K.
(2000). Systematic circular permutation of an entire
protein reveals essential folding elements. Nature
Struct. Biol. 7, 580–585.
43. Lattman, E. E. & Rose, G. D. (1993). Protein folding—
what’s the question? Proc. Natl Acad. Sci. USA, 90,
sub domain isolated from staphylococcal nuclease.
J. Mol. Biol. 250, 134–143.
45. Maity, H., Maity, M., Krishna, M. M., Mayne, L. &
Englander, S. W. (2005). Protein folding: the stepwise
assembly of foldon units. Proc. Natl Acad. Sci. USA,
46. Bhutani, N. & Udgaonkar, J. B. (2003). Folding
subdomains of thioredoxin characterized by native-
state hydrogen exchange. Protein Sci. 12, 1719–1731.
47. Hilser, V. J., Dowdy, D., Oas, T. G. & Freire, E. (1998).
The structural distribution of cooperative interactions
in proteins: analysis of the native state ensemble. Proc.
Natl. Acad. Sci. USA, 95, 9903–9908.
48. Wetlaufer, D. B. (1981). Folding of protein fragments.
Advan. Prot. Chem. 34, 61–92.
49. Lesk, A. M. & Rose, G. D. (1981). Folding units in
globular proteins. Proc. Natl Acad. Sci. USA, 78,
50. Panchenko, A. R., Luthey_Schulten, Z., Cole, R. &
Wolynes, P. G. (1997). The foldon universe: a survey of
structural similarity and self-recognition of indepen-
dently folding units. J. Mol. Biol. 272, 95–105.
51. Tsai, C. J. & Nussinov, R. (1997). Hydrophobic folding
units derived from dissimilar monomer structures
and their interactions. Protein Sci. 6, 24–42.
52. Fischer, K. F. & Marqusee, S. (2000). A rapid test for
proteins. J. Mol. Biol. 302, 701–712.
53. Peng, Z. Y. & Wu, L. C. (2000). Autonomous protein
folding units. Advan. Protein Chem. 53, 1–47.
54. Rohl, C. A., Strauss, C. E., Misura, K. M. & Baker, D.
(2004). Protein structure prediction using Rosetta.
Methods Enzymol. 383, 66–93.
55. Aitken, A. & Learmonth, M. (1996). Performic acid
oxidation. In The Protein Protocols Handbook (Walker,
J. M., ed.), pp. 341–342, Humana Press Inc., Totowa,
56. Ambler, R. P., Coulson, F. W., Frere, J. M., Ghuysen,
J. M., Joris, B., Forsman, M. et al. (1991). A standard
numbering scheme for the class A b-lactamases.
Biochem. J. 276, 269–270.
Edited by C. R. Matthews
(Received 12 December 2005; received in revised form 26 January 2006; accepted 27 January 2006)
Available online 9 February 2006
Conformational Information Throughout Protein Sequence